IL-27 Gene Therapy Ameliorates IPEX Syndrome Caused by Germline Mutation of Foxp3 Gene: A Major Role for Induction of IL-10.

Inactivating mutations of Foxp3, the master regulator of regulatory T cell development and function, lead to immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome in mice and humans. IPEX is a fatal autoimmune disease, with allogeneic stem cell transplant being the only available therapy. In this study, we report that a single dose of adeno-associated virus (AAV)-IL-27 to young mice with naturally occurring Foxp3 mutation (Scurfy mice) substantially ameliorates clinical symptoms, including growth retardation and early fatality. Correspondingly, AAV-IL-27 gene therapy significantly prevented naive T cell activation, as manifested by downregulation of CD62L and upregulation of CD44, and immunopathology typical of IPEX. Because IL-27 is known to induce IL-10, a key effector molecule of regulatory T cells, we evaluated the contribution of IL-10 induction by crossing IL-10-null allele to Scurfy mice. Although IL-10 deficiency does not affect the survival of Scurfy mice, it largely abrogated the therapeutic effect of AAV-IL-27. Our study revealed a major role for IL-10 in AAV-IL-27 gene therapy and demonstrated that IPEX is amenable to gene therapy.

Therapeutic Immunomodulation in Gastric Cancer.

Gastric carcinoma, being one of the most prevalent types of solid tumors, has emerged as the third leading cause of death worldwide. The symptoms of gastric cancer (GC) are typically complex, which makes early detection challenging. Immune checkpoint inhibition has become the new standard targeted therapy for advanced or metastatic GC. It is currently being explored in various combinations, both with and without chemotherapy, across multiple therapies in clinical trials. Immunotherapy can stimulate immune responses in GC patients, leading to the destruction of cancer cells. Compared with traditional therapies, immunotherapy has shown strong effectiveness with tolerable toxicity levels. Hence, this innovative approach to the treatment of advanced GC has gained popularity. In this review, we have outlined the recent advancements in immunotherapy for advanced GC, including immune checkpoint inhibitors, cancer vaccines, vascular endothelial growth factor-A inhibitors, and chimeric antigen receptor T-cell therapy. Our current emphasis is on examining the immunotherapies presently employed in clinical settings, addressing the existing challenges associated with these therapeutic approaches, and exploring promising strategies to overcome their limitations.

CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor-associated myeloid cells.

CD200 is overexpressed in many solid tumors and considered as an immune checkpoint molecule dampening cancer immunity. In this study, we found that CD200R-/- mice were significantly more potent in rejecting these CD200+ tumors. scRNA sequencing demonstrated that tumors from CD200R-/- mice had more infiltration of CD4+ and CD8+ T cells, and NK cells but less infiltration of neutrophils. Antibody depletion experiments revealed that immune effector cells are crucial in inhibiting tumor growth in CD200R-/- mice. Mechanistically, we found that CD200R signaling regulates the expression of chemokines in tumor-associated myeloid cells (TAMCs). In the absence of CD200R, TAMCs increased expression of CCL24 and resulted in increased infiltration of eosinophils, which contributes to anti-tumor activity. Overall, we conclude that CD200R signaling contributes to unfavorable TME through chemokine-dependent recruitment of immune suppressive neutrophils and exclusion of anti-cancer immune effectors. Our study has implications in developing CD200-CD200R targeted immunotherapy of solid tumors.

Neuropeptide Y, a paracrine factor secreted by cancer cells, is an independent regulator of angiogenesis in colon cancer.

BACKGROUND: Resistance to anti-angiogenic therapies targeting vascular endothelial growth factor-A (VEGF-A) stems from VEGF-A independent angiogenesis mediated by other proangiogenic factors. Therefore identifying these factors in colon adenocarcinoma (CA) will reveal new therapeutic targets. METHODS: Neuropeptide Y (NPY) and Y2 receptor (Y2R) expressions in CA were studied by immunohistochemical analysis. Orthotopic HT29 with intact VEGF-A gene and VEGF-A knockdown (by CRISPR/Cas9 gene-editing technique) HT29 colon cancer-bearing mice were treated with specific Y2R antagonists, and the effects on angiogenesis and tumour growth were studied. The direct effect of NPY on angiogenesis and the underlying molecular mechanism was elucidated by the modulation of Y2R receptors expressed on colonic endothelial cells (CEC). RESULTS: The results demonstrated that NPY and Y2R are overexpressed in human CA, orthotopic HT29, and most interestingly in VEGF-A-depleted orthotopic HT29 tumours. Treatment with Y2R antagonists inhibited angiogenesis and thereby HT29 tumour growth. Blocking /silencing Y2R abrogated NPY-induced angiogenic potential of CEC. Mechanistically, NPY regulated the activation of the ERK/MAPK signalling pathway in CEC. CONCLUSIONS: NPY derived from cancer cells independently regulates angiogenesis in CA by acting through Y2R present on CEC. Targeting NPY/Y2R thus emerges as a novel potential therapeutic strategy in CA.

VEGF-A controls the expression of its regulator of angiogenic functions, dopamine D2 receptor, on endothelial cells.

We have previously demonstrated significant upregulation of dopamine D2 (DAD2) receptor (DRD2) expression on tumor endothelial cells. The dopamine D2 receptors, upon activation, inhibit the proangiogenic actions of vascular endothelial growth factor-A (VEGF-A, also known as vascular permeability factor). Interestingly, unlike tumor endothelial cells, normal endothelial cells exhibit very low to no expression of dopamine D2 receptors. Here, for the first time, we demonstrate that through paracrine signaling, VEGF-A can control the expression of dopamine D2 receptors on endothelial cells via Krüppel-like factor 11 (KLF11)-extracellular signal-regulated kinase (ERK) 1/2 pathway. These results thus reveal a novel bidirectional communication between VEGF-A and DAD2 receptors.

Dopamine Prevents Ultraviolet B-induced Development and Progression of Premalignant Cutaneous Lesions through its D2 Receptors.

Although the role of dopamine (DA) in malignant tumors has been reported, its function in premalignant lesions is unknown. Herein we report that the stimulation of DA D2 receptors in endothelial cells in ultraviolet B (UVB)-induced cutaneous lesions in mice significantly reduced the tumor number, tumor burden, and malignant squamous cell carcinoma in these animals. DA D2 receptor agonist inhibited VEGFA-dependent proangiogenic genes in vitro and in vivo. However, the mice pretreated with selective DA D2 receptor antagonist inhibited the actions of the agonist, thereby suggesting that the action of DA was through its D2 receptors in the endothelial cells. To our knowledge, this study is the first to report DA-mediated regulation of pathogenesis and progression of UVB-induced premalignant skin lesions. PREVENTION RELEVANCE: This investigation demonstrates the role of dopamine and its D2 receptors in UVB induced premalignant squamous cell skin lesions and how DA through its D2 receptors inhibits the development and progression of these lesions and subsequently prevents squamous cell carcinoma of the skin.

Suppression of beta 2 adrenergic receptor actions prevent UVB mediated cutaneous squamous cell tumorigenesis through inhibition of VEGF-A induced angiogenesis.

Although beta 2 adrenergic receptors (ß2 ADR) are present in the keratinocytes, their role in cutaneous squamous cell tumorigenesis needs to be ascertained. For the first time, we report here that selective ß2 ADR antagonists by inhibiting ß2 ADR actions significantly retarded the progression of ultraviolet B (UVB) induced premalignant cutaneous squamous cell lesions. These antagonists acted by inhibiting vascular endothelial growth factor-A (VEGF) mediated angiogenesis to prevent UVB radiation-induced squamous cell carcinoma of the skin.

Is AAV-delivered IL-27 a potential immunotherapeutic for cancer?

Cytokines are one of the first immunotherapeutics utilized in trials of human cancers with significant success. However, due to their significant toxicity and often lack of efficacy, cytokines have given their spotlight to other cancer immunotherapeutics such as immune checkpoint inhibitors. Nevertheless, only a subset of cancer patients respond to checkpoint inhibitors. Therefore, developing a novel cytokine-based immunotherapy is still necessary. Among an array of cytokine candidates, IL-27 is a unique one that exhibits clear anti-tumor activity with low toxicity. Systemically delivered IL-27 by adeno-associated virus (AAV-IL-27) is very well tolerized by mice and exhibits potent anti-tumor activity in a variety of tumor models. AAV-IL-27 exerts its anti-tumor activity through directly stimulation of immune effector cells and systemic depletion of Tregs, and is particularly suitable for delivery in combination with checkpoint inhibitors or vaccines. Additionally, AAV-IL-27 can also be delivered locally to tumors to exert its unique actions. In this review, we summarize the evidence that support these points and propose AAV-delivered IL-27 as a potential immunotherapeutic for cancer.

Chebulinic acid is a safe and effective antiangiogenic agent in collagen-induced arthritis in mice.

BACKGROUND: Although vascular endothelial growth factor-A (VEGF)-induced angiogenesis has been reported to play an important role in the pathogenesis of rheumatoid arthritis (RA), serious side effects, mainly grade 2-3 hypertension, which is commonly observed with currently available anti-VEGF agents, can be detrimental for RA patients due to hypertension and associated cardiovascular complications seen in these patients. Thus, identification of anti-VEGF molecules that do not increase blood pressure could be useful for the treatment of RA. Chebulinic acid (CI), a water-soluble small-molecule tannin, can inhibit the actions of VEGF, and a report suggested that CI might not increase blood pressure due to its compensatory effects on the cardiovascular system. Therefore, the effects of CI on blood pressure in mice and the progression of the disease in a murine collagen-induced arthritis (CIA) model were investigated. METHODS: CIA was induced in DBA/1J mice with type II collagen. The effects of CI in these animals were then evaluated by determination of clinical, histopathological, and immunohistochemical parameters. The effects of CI on VEGF-induced proangiogenic genes and signaling pathways were examined in vitro and in vivo. RESULTS: Significant CD31 and VEGF expressions were detected in the synovial tissues of mice with CIA, similar to their expressions observed in human RA patients. However, treatment with CI significantly inhibited paw swelling, decreased the mean articular index and joint pathology scores in these animals through inhibition of VEGF-induced proangiogenic gene expressions and signaling pathways that regulate angiogenesis. Unlike currently used antiangiogenic agents, CI at a dose that inhibits VEGF actions did not increase blood pressure in mice. CONCLUSION: CI can act as a safe and potent anti-VEGF antiangiogenic agent for the treatment of types of inflammatory arthritis, such as RA.

Angiogenesis Inhibition in Prostate Cancer: An Update.

Prostate cancer (PCa), like all other solid tumors, relies on angiogenesis for growth, progression, and the dissemination of tumor cells to other parts of the body. Despite data from in vitro and in vivo preclinical studies, as well as human specimen studies indicating the crucial role played by angiogenesis in PCa, angiogenesis inhibition in clinical settings has not shown significant benefits to patients, thus challenging the inclusion and usefulness of antiangiogenic agents for the treatment of PCa. However, one of the apparent reasons why these antiangiogenic agents failed to meet expectations in PCa can be due to the choice of the antiangiogenic agents, because the majority of these drugs target vascular endothelial growth factor-A (VEGFA) and its receptors. The other relevant causes might be inappropriate drug combinations, the duration of treatment, and the method of endpoint determination. In this review, we will first discuss the role of angiogenesis in PCa growth and progression. We will then summarize the different angiogenic growth factors that influence PCa growth dynamics and review the outcomes of clinical trials conducted with antiangiogenic agents in PCa patients and, finally, critically assess the current status and fate of antiangiogenic therapy in this disease.

Activation of D2 Dopamine Receptors in CD133+ve Cancer Stem Cells in Non-small Cell Lung Carcinoma Inhibits Proliferation, Clonogenic Ability, and Invasiveness of These Cells.

Lung carcinoma is the leading cause of cancer-related death worldwide, and among this cancer, non-small cell lung carcinoma (NSCLC) comprises the majority of cases. Furthermore, recurrence and metastasis of NSCLC correlate well with CD133+ve tumor cells, a small population of tumor cells that have been designated as cancer stem cells (CSC). We have demonstrated for the first time high expression of D2 dopamine (DA) receptors in CD133+ve adenocarcinoma NSCLC cells. Also, activation of D2 DA receptors in these cells significantly inhibited their proliferation, clonogenic ability, and invasiveness by suppressing extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT, as well as down-regulation of octamer-binding transcription factor 4 (Oct-4) expression and matrix metalloproteinase-9 (MMP-9) secretion by these cells. These results are of significance as D2 DA agonists that are already in clinical use for treatment of other diseases may be useful in combination with conventional chemotherapy and radiotherapy for better management of NSCLC patients by targeting both tumor cells and stem cell compartments in the tumor mass.

Activation of Dopamine D1 Receptors in Dermal Fibroblasts Restores Vascular Endothelial Growth Factor-A Production by These Cells and Subsequent Angiogenesis in Diabetic Cutaneous Wound Tissues.

In wound beds, fibroblasts are rich sources of vascular endothelial growth factor A, a cytokine necessary for promoting angiogenesis and thereby the healing of wound tissues. However, in diabetes mellitus, these cells are functionally impaired and produce reduced amounts of vascular endothelial growth factor A, resulting in deficient angiogenesis and delayed wound healing. We here for the first time demonstrate that stimulation of D1 dopamine receptors present in dermal fibroblasts restores vascular endothelial growth factor A production by these cells, resulting in adequate angiogenesis and subsequent healing of cutaneous wounds in both type 1 and type 2 diabetic mice. This action of D1 dopamine receptors was mediated through the protein kinase A pathway. As delayed wound healing or chronic wounds are one of the major health problems in diabetic patients, D1 dopamine receptor agonists, which are already in clinical use for the treatment of other disorders, may be of translational value in the treatment of chronic, nonhealing diabetic wounds.

A Critical Role for CD200R Signaling in Limiting the Growth and Metastasis of CD200+ Melanoma.

CD200 is a cell surface glycoprotein that functions through engaging CD200R on cells of the myeloid lineage and inhibits their functions. Expression of CD200 was implicated in a variety of human cancer cells, including melanoma cells; however, its roles in tumor growth and immunity are not clearly understood. In this study, we used CD200R-deficient mice and the B16 tumor model to evaluate this issue. We found that CD200R-deficient mice exhibited accelerated growth of CD200(+), but not CD200(-), B16 tumors. Strikingly, CD200R-deficient mice receiving CD200(+) B16 cells i.v. exhibited massive tumor growth in multiple organs, including liver, lung, kidney, and peritoneal cavity, whereas the growth of the same tumors in wild-type mice was limited. CD200(+) tumors grown in CD200R-deficient mice contained higher numbers of CD11b(+)Ly6C(+) myeloid cells, exhibited increased expression of VEGF and HIF1α genes with increased angiogenesis, and showed significantly reduced infiltration of CD4(+) and CD8(+) T cells, presumably as the result of reduced expression of T cell chemokines, such as CXCL9 and CXCL16. The liver from CD200R-deficient mice, under metastatic growth of CD200(+) tumors, contained significantly increased numbers of CD11b(+)Gr1(-) myeloid cells and Foxp3(+) regulatory T cells and reduced numbers of NK cells. Liver T cells also had a reduced capacity to produce IFN-γ or TNF-α. Taken together, we revealed a critical role for CD200R signaling in limiting the growth and metastasis of CD200(+) tumors. Thus, targeting CD200R signaling may potentially interfere with the metastatic growth of CD200(+) tumors, like melanoma.

Plants and their active compounds: natural molecules to target angiogenesis.

Angiogenesis, or new blood vessel formation, is an important process in the pathogenesis of several diseases and thus has been targeted for the prevention and treatment for many disorders. However, the anti-angiogenic agents that are currently in use are mainly synthetic compounds and humanized monoclonal antibodies, which are either expensive or toxic, thereby limiting their use in many patients. Therefore, it is necessary to identify less toxic, inexpensive, novel and effective anti-angiogenic molecules. Several studies have indicated that natural plant products can meet these criteria. In this review, we discuss the anti-angiogenic properties of natural compounds isolated from plants and the molecular mechanisms by which these molecules act. Finally, we summarize the advantages of using plant products as anti-angiogenic agents. Compared with currently available anti-angiogenic drugs, plant products may not only have similar therapeutic potential but are also inexpensive, less toxic, and easy to administer. However, novel and effective strategies are necessary to improve their bioavailability for clinical use.

The natural compound chebulagic acid inhibits vascular endothelial growth factor A mediated regulation of endothelial cell functions.

Vascular endothelial growth factor A (VEGFA) plays an important role in tumour angiogenesis and its angiogenic action is mainly mediated through its VEGF receptor 2 (VEGFR-2). Therefore drugs targeting VEGFA/VEGFR-2 are being presently used in the clinics for treatment of several types of solid malignant tumours. We here in report that low dose of chebulagic acid (CA), a hydrolysable tannin found in myrobalan fruits can inhibit VEGFA induced vascular permeability, endothelial cell proliferation, migration, tube formation and thereby, angiogenesis by suppressing VEGFR-2 phosphorylation. CA may thus be an effective and useful natural inhibitor of VEGFA mediated angiogenesis.

Dopamine is a safe antiangiogenic drug which can also prevent 5-fluorouracil induced neutropenia.

The role of vascular endothelial growth factor A (VEGFA) in tumor angiogenesis is well established and accordingly, molecules targeting VEGFA or its receptors are being presently used in the clinics for treatment of several types of cancer. However, these antiangiogenic agents are expensive and have serious side effects. Thus identification of newer drugs with manageable systemic side effects or toxicities is of immense clinical importance. Since we have reported earlier that dopamine (DA) inhibits VEGFA induced angiogenesis in experimental tumor models, we therefore sought to investigate whether DA treatment results in similar toxicities like other antiangiogenic agents. Our results indicated that unlike sunitinib, another commonly used antiangiogenic agent in the clinics which targets VEGF receptors, DA [50 mg/kg/days × 7days intraperitoneally (i.p.)] not only could inhibit tumor angiogenesis and growth of HT29 human colon cancer and LLC (Lewis lung carcinoma) in mice, it also did not cause hypertension, hematological, renal and hepatic toxicities in normal, HT29 and LLC tumor bearing animals. Furthermore and interestingly, in contrast to the currently used antiangiogenic agents, DA also prevented 5-fluorouracil (5FU) induced neutropenia in HT29 colon cancer bearing mice. This action of DA was through inhibition of 5FU mediated suppression of colony forming unit-granulocyte macrophage colony forming units in the bone marrow. Thus our results indicate that DA may be safely used as an antiangiogenic drug for the treatment of malignant tumors.

Nanofiber-expanded human umbilical cord blood-derived CD34+ cell therapy accelerates murine cutaneous wound closure by attenuating pro-inflammatory factors and secreting IL-10.

Nanofiber-expanded human umbilical cord blood-derived CD34+ cell therapy is under consideration for treating peripheral and cardiac ischemia. However, the therapeutic efficacy of nanofiber-expanded human umbilical cord blood-derived (NEHUCB) CD34+ cell therapy for wound healing and its mechanisms are yet to be established. Using an excision wound model in NOD/SCID mice, we show herein that NEHUCB-CD34+ cells home to the wound site and significantly accelerate the wound-healing process compared to vehicle-treated control. Histological analysis reveals that accelerated wound closure is associated with the re-epithelialization and increased angiogenesis. Additionally, NEHUCB-CD34+ cell-therapy decreases expression of pro-inflammatory cytokines, such as TNF-α, IL-1ß, IL-6 and NOS2A in the wound bed, and concomitantly increases expression of IL-10 compared to vehicle-treated control. These findings were recapitulated in vitro using primary dermal fibroblasts and NEHUCB-CD34+ cells. Moreover, NEHUCB-CD34+ cells attenuate NF-κB activation and nuclear translocation in dermal fibroblasts through enhanced secretion of IL-10, which is known to bind to NF-κB and suppress transcriptional activity. Collectively, these data provide novel mechanistic evidence of NEHUCB-CD34+ cell-mediated accelerated wound healing.

Knockdown of ß-catenin with dicer-substrate siRNAs reduces liver tumor burden in vivo.

Despite progress in identifying molecular drivers of cancer, it has been difficult to translate this knowledge into new therapies, because many of the causal proteins cannot be inhibited by conventional small molecule therapeutics. RNA interference (RNAi), which uses small RNAs to inhibit gene expression, provides a promising alternative to reach traditionally undruggable protein targets by shutting off their expression at the messenger RNA (mRNA) level. Challenges for realizing the potential of RNAi have included identifying the appropriate genes to target and achieving sufficient knockdown in tumors. We have developed high-potency Dicer-substrate short-interfering RNAs (DsiRNAs) targeting ß-catenin and delivered these in vivo using lipid nanoparticles, resulting in significant reduction of ß-catenin expression in liver cancer models. Reduction of ß-catenin strongly reduced tumor burden, alone or in combination with sorafenib and as effectively as DsiRNAs that target mitotic genes such as PLK1 and KIF11. ß-catenin knockdown also strongly reduced the expression of ß-catenin-regulated genes, including MYC, providing a potential mechanism for tumor inhibition. These results validate ß-catenin as a target for liver cancer therapy and demonstrate the promise of RNAi in general and DsiRNAs in particular for reaching traditionally undruggable cancer targets.

Tumor-derived IL-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis.

IL-35 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune-suppressive activity. Although IL-35 was demonstrated to be produced by regulatory T cells, gene-expression analysis revealed that it is likely to have a wider distribution, including expression in cancer cells. In this study, we demonstrated that IL-35 is produced in human cancer tissues, such as large B cell lymphoma, nasopharyngeal carcinoma, and melanoma. To determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35-producing plasmacytoma J558 and B16 melanoma cells and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but it stimulates tumorigenesis in both immune-competent and Rag1/2-deficient mice. Tumor-derived IL-35 increases CD11b(+)Gr1(+) myeloid cell accumulation in the tumor microenvironment and, thereby, promotes tumor angiogenesis. In immune-competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor Ag-specific CD8(+) T cell activation, differentiation, and effector functions. However, IL-35-treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions for IL-35 in promoting tumor growth via the enhancement of myeloid cell accumulation, tumor angiogenesis, and suppression of tumor immunity.